Improved Method to Study Protein-DNA Interactions

CUT&RUN

CUT&RUN, which stands for cleavage under targets and release using nuclease offers a new approach to pursue epigenetics1. CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP (Fig 1), making CUT&RUN especially cost-effective for transcription factor and chromatin profiling1.

 Figure 1: Superior Peak Calling with CUT&RUN (green) in Comparison to ChIP-seq (orange)

However it is not limited to profiling; CUT&RUN has the potential to replace all ChIP-based applications.

Note: antibodies-online™ CUT&RUN sets are now available! Discover the different compilations tailored to your needs Get started now!

Discover all antibodies-online™ CUT&RUN Sets

Table. 1: CUT&RUN Sets available. Contact our support for detailed information on each set.
Positive & Negative Control Rabbit anti-Mouse Secondary Rabbit anti-DYKDDDDK Primary Mouse anti-DYKDDDDK Primary ConA Beads
CUT&RUN Pro Complete ABIN6923135
CUT&RUN Pro Direct ABIN6923136
CUT&RUN Pro Sec ABIN6923137
CUT&RUN Pro ABIN6923138
CUT&RUN Core Complete ABIN6923131
CUT&RUN Core Direct ABIN6923132
CUT&RUN Core Sec ABIN6923133
CUT&RUN Core ABIN6923134
Image
Better Data
Only 1/3 of seq reads required
Image
Less Signal Noise
Easer Peak-Calling
Higher Reproducibility
Image
Less Sample
10x less sample in comparison to ChIP-seq
Image
Optimized Protocol
Obtain purified DNA from Cells within 1 day

CUT&RUN - Combining Perks of ChIP‐Seq Variations

As CUT&RUN is performed on intact cells or nuclei without fragmentation, it can be used to probe all genomic compartments. The cleaved chromatin complexes diffuse out of the nuclei where they can be harvested in supernatant. The rest of the undigested genome is retained in the intact nuclei (Fig 3). For ChIP-Seq on the other hand, the majority of chromatin is sheared or digested resulting in comparison in a worse signal-to-noise ratio (Fig 3-5). Consequently, ChIP-Seq requires more sequencing depth and with each sequencing run additional sample amount, labour time and money.

Extraction without fragmentation

The CUT&RUN is straightforward and can be completed in under a day using standard lab equipment. CUT&RUN is suitable for application down to 100 cells for profiling H3K27me3 or 1000 cells for CTCF sequence-specific DNA- binding protein 1. Therefore, CUT&RUN enables targeted genome-wide maps of protein- DNA interactions even for rare cell types. In comparison with XChiP-seq data (orange) both CUT&RUN runs (green) reveal reliable peaks with less background noise (Fig 1). One of the strengths of CUT&RUN is that the entire reaction is performed in situ.

In combination with a proximity ligation assay the generation of factor-specific high resolution maps of nuclear architecture is possible.

Experimental Design

Figure 2: CUT&RUN Diffusion

Fig.4.: Extraction without fragmentation.

  1. AB and Prot-A-MNase Fusion Protein diffuse in.
  2. Protein-DNA Complex diffuses into solution.

CUT&RUN

Figure 3: CUT&RUN

Native ChIP

Figure 4: Native ChIP (NChIP)

Cross-Linked ChIP

Figure 5: Cross-Linked ChIP

Importance of Antibody Selection

The successful execution of cleavage under target and release using nuclease hinges on antibody selection. A factor- or histone-specific antibody is bound to chromatin in situ followed by binding to the antibody of a protein A-micrococcal nuclease (pA-MNase) fusion. As is the case with ChIP, the success depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. The following list serves as an example, Brahma and Henikoff successfully utilized this compilation in 20182.

Note: You need help to choose the fitting antibody for your experiment? Our tech support will assist you!

CUT&RUN Protocol Steps

  1. Hypotonic Lysis to release Nuclei
  2. Imobilize Nuclei with Magnetic Beads
  3. Incubate with Antibody against PoI
  4. Incubate with ProteinA-Mnase
  5. Add Ca+2 (Reaction Start)
  6. Add Chelator (Reaction Stop)
  7. Pellet oligonucleosome
  8. Sequencing
Note: Contact us in order to get the full CUT&RUN protocol!

Single Set Components available

Suggested reagents based on Brahma et al.

Workflow

Image

Frequently Asked Questions

Why is the DNA yield so low?

CUT&RUN is performed using low cell numbers and the background signal is considerable lower than e.g. for ChIP. Due to these two factors the amount of recovered DNA is often times too low to be reliably measured based on a fluorometric assay or by capillary electrophoresis. PCR amplification of small CUT&RUN products, i.e. less than 50 bp, can be problematic and is therefore not any option.

In order to assess the success of the CUT&RUN method each of the antibodies-online CUT&RUN product sets includes the CUT&RUN Positive Control antibody ABIN6923144 against the abundant H3K27me3 histone modification. DNA fragments prepared using this antibody can be measured by sensitive electrophoresis on a Bioanalyzer or Tapestation or fluorometrically on a Qubit or Nanodrop fluorometer. When using the CUT&RUN Positive Control antibody ABIN6923144 (or any other antibody specific for nucleosomal markers) a ladder corresponding to multiples of the 147 bp long nucleosomes should be visible by capillary electrophoresis.

Why do I need a negative control antibody? Why not just use a no-antibody control?

MNase is an endo- and exonuclease that will unspecifically bind and cleave unprotected DNA in hyper-accessible DNA, e.g. in regions surrounding regulatory elements. Free MNase will preferentially cut DNA within these hyper-accessible regions, thus potentially causing false positives and increased background signal in general.

In order to avoid this undesired effect of untethered MNase, the chromatin is randomly coated with the CUT&RUN Negative Control ( ABIN6923140) prior to the addition of pA/G-MNase is added to the samples. pA/G-MNase is then tethered via its Protein A or Protein G portion to the antbodies’ Fc fragment and background DNA fragmentation is dictated by the random antibody binding as opposed to the nuclease digestion of hyper-accessible DNA regions.

Are there protocols to make this work for tissue, where I cannot necessarily isolate single cells?

The Henikoff lab that published the original CUT&RUN method in 2017 has since developed several variations. One of these modification is AutoCUT&RUN which outlines a workflow to automate CUT&RUN for high-throughput characterization of single cells but also patient samples and xenografts.

You can find the detailed protocol at protocols.io.

In short, the tissue is processed manually or enyzmatically into single, intact cell that are subsequently bound to ConA beads. The improved (and standard) CUT&RUN protocol that serve as basis for the protocol outlined in our CUT&RUN handbook can be adapted accordingly. Presently there is no work published documenting the use of intact tissue.

Is it possible to use the CUT&RUN product sets with plant tissue samples?

The CUT&RUN method can be applied without a problem to plant tissue samples (see e.g. PMID30719569). One essential step in addition to those lined out in the protocol is the generation of spheroblasts so that it becomes possible to permeabilize the plasma membrane for the application of the antibodies and the MNase fusion protein.

The positive control H3K27me3 antibody and the negative control guinea pig anti-rabbit IgG antibody as well as the ConA beads are suitable for use with plant samples. The antibodies that are included in some of the sets, such as the anti-DYKDDDDK antibodies or the secondary antibodies, can also be used with plant samples. Whether they are needed or not depends on your experiment.

Can CUT&RUN be adapted for RIP-seq?

It should be possible to adapt the CUT&RUN protocol for use with RNA as an alternative to RIP-seq. RNA in the cytoplasm will attract the degradation machinery if it is lacking the 5' cap and the 3' poly-A tail. Thus, it is advisable to use isolated nuclei as sample material. Consequently, no digitonin in the different buffers is needed since the nuclear envelope does not contain cholesterol. Isolated nuclei may be immobilized via glycoproteins on the nuclear envelope to the ConA beads like for a CUT&RUN experiment. An antibody against the protein of interest is added and subsequently the pA/G-MNase is tethered to the antibody, thus bringing the MNase into proximity of the RNA of interest. Similar to RIP-seq, isolated RNA is then translated into cDNA and can be sequenced and mapped.

Is it possible to use a cell free extract of a bacterial cell instead of immobilizing and permeabilizing the cell?

It should indeed be possible to use a bacterial extract as starting material instead of immobilized eukaryotic cells. There are a couple of things that should be considered:

One of the factors that makes CUT&RUN so much better than ChIP-seq in a eukaryotic system is that it is done in situ. The intact cells (or isolated nuclei) retain the large, undigested chromatin. Consequently, much fewer off-sites are present in the prepared DNA which is one of the reasons why CUT&RUN data sets have so much less background signal when compared to ChIP-seq data. This enrichment step will be missing when using a cell lysate as starting material. The CUT&RUN result should still be superior to the CHIP-seq result for the same starting material. However, it will be more "noisy" than CUT&RUN sequencing data from intact eukaryotic cells/nuclei.

The buffers described in the CUT&RUN protocol are designed to maintain the cells in the samples intact. The binding buffer contains some divalent cations that are necessary for the binding of the cells to the Concanavalin on the beads that are used for the immobilization. Subsequently, this buffer is changed for an antibody buffer that contains EDTA to chelate the divalent cations and prevent DNA cleavage prior to pA/G-MNase antibody binding. Since you are working with cell lysates as opposed to intact cells there is no need to include digitonin in any of the buffers. You don't need the binding buffer used to immobilize cells on the ConA beads. In order to control premature DNA cleavage by the MNase I would include some EDTA and/or EGTA in the lysis buffer.

Probably, you will have to titrate the amount of pA/G-MNase and try different digestion times to find the best balance between efficient cleavage of the intended sites and too much off-site cleavage.

The original CUT&RUN protocol foresees the use of heterologous spike-in DNA, e.g. from E. coli. In its latest iteration it is argued that there is no need to add this DNA since the pA/G-MNase preparation contains sufficient levels of E. coli DNA to serve as a standard for quantitation. In a prokaryotic system, spike-in DNA from a eukaryotic organism (e.g. S. cerevisiae) is recommended. Also keep in in mind that the E. coli DNA carried over with the pA/G-MNase preparation may contain sequences that show homology to the bindings sites of your protein of interest.

Our product sets are optimized for use with eukaryotic cells. The positive control in all sets is a recombinant rabbit anti-H3K27me3 antibody. This control is not suitable for use with prokaryotic samples. Get in touch with us at mail to find the necessary reagents for your experiment.


References:

  1. Peter J. Skene and Steven Henikoff (2018): "CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers”. Nature, Volume 13, pages 1006–1019. [PMID: 25652980]
  2. Sandipan Brahma and Steven Henikoff (2018): "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast". Mol Cell, Volume 73, Issue2, P238-249. [DOI]

KEYWORDS:
Cut and Run, CutAndRun, ChIP-seq, ChIP-sequencing, chromatin; chromosomes; epigenomics; gene expression; genetics; genomics; human; spike-in calibration; in situ profiling; transcription factors; A-Micrococcal Nuclease